ROLE OF THE INTERGENIC DINUCLEOTIDE IN VESICULAR STOMATITIS-VIRUS RNA-TRANSCRIPTION

To investigate the role played by the intergenic dinucleotide sequence of the conserved vesicular stomatitis virus (VSV) gene junction in mo dulation of polymerase activity, we analyzed the RNA synthesis activit ies of bicistrionic genomic analogs that contained either the authenti c N/P gene junction or gene junctions that had been altered to contain either the 16 possible dinucleotide combinations, single nucleotide i ntergenic sequences, or no intergenic sequence at all. Quantitative me asurements of the amounts of upstream, downstream, and readthrough mRN As that were transcribed by these mutant templates showed that the beh avior of the viral polymerase was profoundly affected by the nucleotid e sequence that it encountered as it traversed the gene junction, alth ough the polymerase was able to accommodate a remarkable degree of seq uence variation without altogether losing the ability to terminate and reinitiate transcription. Alteration or removal of the intergenic seq uence such that the U tract responsible for synthesis of the upstream mRNA poly(A) tail was effectively positioned adjacent to the consensus downstream gene start signal resulted in almost complete abrogation o f downstream mRNA synthesis, thus defining the intergenic sequence as an essential sequence element of the gene junction. Many genome analog s with altered intergenic sequences directed abundant synthesis of a r eadthrough transcript without correspondingly high levels of downstrea m mRNA, an observation inconsistent with the shunting model of VSV tra nscription, which suggests that polymerase molecules are prepositioned at gene junctions, awaiting a push from upstream. Instead, the findin gs of this study support a model of sequential transcription in which initiation of downstream mRNA can occur only following termination of the preceding transcript.